A high-throughput assay for the comprehensive profiling of DNA ligase fidelity

Abstract

DNA ligases have broad application in molecular biology, from traditional cloning methods to modern synthetic biology and molecular diagnostics protocols. Ligation-based detection of polynucleotide sequences can be achieved by the ligation of probe oligonucleotides when annealed to a complementary target sequence. In order to achieve a high sensitivity and low background, the ligase must efficiently join correctly base-paired substrates, while discriminating against the ligation of substrates containing even one mismatched base pair. In the current study, we report the use of capillary electrophoresis to rapidly generate mismatch fidelity profiles that interrogate all 256 possible base-pair combinations at a ligation junction in a single experiment. Rapid screening of ligase fidelity in a 96-well plate format has allowed the study of ligase fidelity in unprecedented depth. As an example of this new method, herein we report the ligation fidelity of Thermus thermophilus DNA ligase at a range of temperatures, buffer pH and monovalent cation strength. This screen allows the selection of reaction conditions that maximize fidelity without sacrificing activity, while generating a profile of specific mismatches that ligate detectably under each set of conditions.

Kinetic mechanism and fidelity of nick sealing byEscherichia coliNAD+-dependent DNA ligase (LigA)

“…Fidelity refers to the capacity of a ligase to discriminate in the sealing of substrates containing paired versus mispaired bases flanking the nick. Different DNA ligases display distinct patterns of fidelity, with respect to how well the ligase discriminates and the hierarchy of which mispairs are or are not accepted by the ligase ( 38 – 49 ). Here we evaluated the fidelity of Eco LigA, focusing first on the effects of 5′-PO 4 base mispairs on the transient state kinetics of nick sealing.…”

Cyclization of secondarily structured oligonucleotides to single-stranded rings by using Taq DNA ligase at high temperatures

“…For example, a splint of 40 nt or longer was oen employed for the ligation at 60 C or lower. [36][37][38] However, such long splints cannot be used for selective DNA ring production, since they cover a large portion of l-DNAs and the cyclic intermediates are hard to be formed due to higher rigidity, especially for a short l-DNA. Very recently, however, we have unexpectedly found that much shorter splint is sufficiently effective for this enzymatic ligation at 65 C ( Fig.…”

Adapting capillary gel electrophoresis as a sensitive, high-throughput method to accelerate characterization of nucleic acid metabolic enzymes

“…We provide examples that illustrate how assays can be adapted for high-throughput CE analysis of a range of nucleic acid enzymes. The accompanying manuscript describes in detail how the high-throughput CE assay can be used to systematically and comprehensively characterize DNA ligase kinetics and fidelity ( 25 ).…”